Influenza A viruses are responsible for the major pandemics of influenza in the last century and are also the causative agents for most of the annual outbreaks of epidemic influenza. The influenza virus is an enveloped virus belonging to the family of the Orthomyxoviridae. Within the envelope, the virus carries eight different RNA segments. There are many different influenza strains, and based on distinctive immunogenic properties, the virus are classified into three types: influenza A, B and C viruses (Influenza. Kilbourne E D ed. Plenum press 1987).
Influenza virus carries at least two different surface glycoprotein antigens on the external envelop, hemagglutinin (HA) trimer, consisting of three individual HA monomers, and the neuraminidase (NA) that exists as tetramer. Both HA and NA plays pivotal role during infection into susceptible cells. There are very immunogenic and elicits specific antibody responses during infection.
There are many different influenza A virus subtypes, differing in the nature of the HA and NA glycoproteins on their surface. Sixteen HAs (H1 to H16) and nine NAs (N1 to N9) have been identified. Among these, H1, H2 and H3 virus subtypes have been identified in humans, specifically the H1N1, H2N2 and H3N2 viruses corresponding to the three major pandemics of the last century (Fields Virology 4th, ed. Fields B N, Knipe D M and Howly P M eds, 1489, 2001). Virus subtypes are distinguishable serologically, which means that antibodies to one subtype do not properly react with another subtype. Besides, humans, influenza viruses infect variety of hosts including swine, avian and equestrian species. Among these, aquatic birds appear to serve as a major reservoir of influenza A viruses; indeed, all human influenza virus subtypes circulate also in these wild birds. During resent outbreaks of avian influenza, there have been occasional transmissions of H5N1, H7N7 and H9N2 viruses to humans (Proc. Natl. Acad. Sci. USA 101, 8156-8161, 2004).
Besides influenza A subtypes, influenza B virus also infects humans especially very young children. Serologically, influenza B virus is clearly distinct from two different subtypes of influenza A viruses (A/H1N1 and A/H3N2) that currently circulates globally. For this reason, current influenza vaccine stipulates the use of three different components (A/H1N1, A/3N2 and B virus) as trivalent vaccine formula.
Because of high mutation rate of the RNA genome, different strains emerge almost every year that cause annual influenza epidemics. Antigenic drift involves minor changes in the RA, NA and possibly also other viral antigen, that occur due to mutations in the viral genome, resulting in amino acid substitution in antigenic sites. These changes may render the new strain different enough to at least partially avoid the immunity induced by previous strains.
Since influenza virus genomes are segmented, influenza virus frequently undergoes more drastic changes ‘antigenic shift’ by genetic reassortment between different viruses. This means that a virus with a new HA (and NA) are introduced into the human population. Especially among those who are immunologically naïve, the infection would spread rapidly and cause high morbidity and mortality among the entire population, including young healthy people. Human population has experienced at least three major influenza pandemics in the 20th century. The Spanish influenza (caused by an H1N1, influenza A virus) in 1918 resulted in the death to 20 to 50 million worldwide. The sequence of the virus shows that similar mutations were also observed in the recent H5N1 avian influenza isolates that infects humans (Nature 437, 889-93, 2005). Other pandemics occurred in 1957 (Asian flu, 112N2) and 1968 (Hong Kong flu, H3N2) with a total number of deaths of approximately 2 million. Moreover, there are numerous reports on human infection by avian influenza viruses (including H5N1, H7N7 and H9N2 viruses) in the past several years. The current information supports the concept that new pandemic influenza is derived from avian virus reservoirs. Avian influenza viruses may be directly transmitted to humans, which probably occurred in the case of the 1918 Spanish flu virus. The possibility of direct transmission of an avian influenza virus to humans became evident for the first time during the H5N1 outbreak in Hong Kong in 1997.
Recently, influenza B viruses have been isolated from animals, demonstrating that influenza B viruses are not restricted to humans and raising concerns about the potential for influenza B viruses to emerge with new antigenic properties. Moreover, influenza B virus infection has been associated with acute necrotizing encephalopathy (ANE) and influenza B-associated encephalitis (IBAE) and neurological sequela.
Vaccination remains the cornerstone of influenza prevention, and currently, trivalent vaccine containing three different influenza surface antigens (A/H1N1, A/H3N2 and B virus) are being used. Antigenic drift of established human virus subtypes requires regular update of the composition of the annual influenza vaccine necessitating annual vaccination. For this reason, current influenza vaccine stipulates the use of three different components as trivalent vaccine formula. In addition, because of potential circulation among humans of avian influenza viruses such as H5N1, H9N2 or H7N7, tetravalent and even multivalent vaccine can also be used for cross-protection against various influenza viruses.
There are several potential strategies for the development of vaccines to protect humans against influenza viruses, including (1) inactivated vaccine; (2) subunit vaccine that uses purified HA and NA components; and (3) live attenuated vaccines. Live attenuated vaccines generally provide better protection than inactivated on subunits vaccines as exemplified by previous developed A/Ann Arbor/6/60 (AA) (H2N2) virus or A/Lenhngrad/134/47/57 (H2N2) (Antiviral Res. 1, 339-365, 1981). The live vaccine stimulates the secretion of IgA class antibodies in the respiratory tract and inactivates the infecting virus on the infection site providing an on-site protection. (New Eng J Med. 338, 1405-1412, 1998; Vaccine 18, 82-88, 2000)
Classically, live attenuated vaccine has been developed through cold adaptation of infectious viruses (Antiviral Res. 1, 339-365, 1981). Using this virus as backbone strain, recombinant virus could be generated by annual reassortment between the ca virus and seasonal virulent influenza virus. The reassortant virus—carrying the surface antigens (RA ad NA) derived from the virulent strain and the six internal RNAs inherited from the ca virus—is immunologically identical to virulent strain but as attenuated as the parental ca virus. The virus, when given to human preferably through nasal route, does not cause virulent symptoms, and yet would induce specific protective immune response. Influenza vaccines are generally produced from virus grown on embryonated chicken eggs.
Ideally, a live vaccine should be both safe, effective in providing protection and cost-effective in production. For safety, the vaccine should be sufficiently attenuated and would not cause clinical symptom even at high dose of vaccination. For effective protection, the vaccine, even at low dose of vaccination, should be immunogenic enough to provide sufficient protection from virulent infection. Moreover, the vaccine strain must retain good growth property in embryonated eggs to reduce the production cost. The current global production capacity of egg-based influenza vaccine is very limited, with only 0.3 to 0.5 billion doses annually, which covers only 6-10% of global population. In time of pandemics (H5N1 avian influenza, for example), the whole population is expected to suffer from the shortage of vaccine supply. Therefore, high-titre production of in embryonated eggs is especially important for influenza vaccine (WHO/CDS/CSR/RMD/2004.8).
In practice, however, it is usually difficult to generate a vaccine strain that satisfies all three requirements-safety, efficacy and the cost-effectiveness. This difficulty is inherently associated with the process of attenuation and the use of live vaccine. Usually, attenuation (providing safety) results in poor growth (at the expense of cost-effectiveness), and renders the virus less immunogenic (requiring high dose of vaccination). In addition, since there are many variants of influenza viruses, matching of surface antigens (HA and NA) between the vaccine and the virulent virus is really important for protection. Therefore, prediction of influenza, strains should be made well ahead of actual circulation of seasonal influenza in winter time. If prediction fails, the vaccine of that particular influenza season is not effective. Unfortunately, the current inactivated influenza vaccine relies on specific, delayed immune response and does not provide enough cross-protection to different subtypes of influenza viruses (J Am Med Assoc 253, 1136-1139, 1985). Moreover, the vaccine does not provide protection from immediate infection (within one to five days before infection, for example), and should be given well ahead of infection, preferably at least 24 weeks before infection, to provide good protection. In contrast to seasonal influenza, the outbreak of avian influenza infection cannot be predicted (Cell 124: 665-70, 2006). The current influenza vaccine therefore has intrinsic limitation in providing immediate protection against sudden influenza outbreaks with unknown identity.
As preparedness for newly emerging threat posed by avian influenza, WHO now recommends, in addition to stockpiling of antivirals, the development of pandemic vaccines suitable for mass immunization administered in non-invasive route (WHO/CDS/CSR/RMD/2004.8). Although vaccines and antiviral drugs constitute essential components for this purpose, they present intrinsic limitations as efficient control measures of pandemics: antivirals such as Tamiflu™ are usually effective only after infection whereas vaccines should be administered well ahead of infection, preferably at least 2 to 4 weeks before exposure to circulating viruses: Currently, there is no effective means for controlling immediate infection. This issue is important especially for avian influenza, where outbreak and the speed of spread among human population are unpredictable.
The limitation of the current influenza vaccine therefore calls for the development of novel vaccine and prophylactic strategies. Ideally, an efficient vaccine would provide both (1) broad-spectrum immediate prophylaxis against variety of influenza viruses of unknown identity, and (2) delayed but specific protection against circulating virus of which the identity has been predicted when used well ahead of infection, preferably 2-4 weeks before infection following generally recommended vaccination schedule.
Accordingly, the development of novel live influenza vaccine that addresses to all important issues: safety, efficacy, high production yield, immediate protection against variety of influenza subtypes and prolonged protection against specific influenza subtype is needed.